RESSALVA Atendendo solicitação do autor, o texto completo desta tese será disponibilizado somente a partir de 03/04/2026 RENAN PEREIRA PEDRO ABORDAGEM DUAL NA INVESTIGAÇÃO DA PROTEÍNA GRB2: EXPLORAÇÃO DA INTERAÇÃO ESPECÍFICA E ANÁLISE DINÂMICA DO ENOVELAMENTO São José do Rio Preto 2024 1 RENAN PEREIRA PEDRO ABORDAGEM DUAL NA INVESTIGAÇÃO DA PROTEÍNA GRB2: EXPLORAÇÃO DA INTERAÇÃO ESPECÍFICA E ANÁLISE DINÂMICA DO ENOVELAMENTO Tese apresentada como parte dos requisitos para obtenção do título de Doutor em Biofísica Molecular, junto ao Programa de Pós Graduação em Biofísica Molecular do Instituto de Biociências, Letras e Ciências Exatas da Universidade Estadual Paulista “Júlio de Mesquita Filho”, Câmpus de São José do Rio Preto. Financiadora: CNPq - Processo: 140306/2020-0 Orientador: Prof. Dr. Fernando Alves de Melo Coorientador: Prof. Dr. Ícaro Putinhon Caruso Coorientador: Dr. Raphael Vinícius Rodrigues Dias São José do Rio Preto 2024 1 2 RENAN PEREIRA PEDRO ABORDAGEM DUAL NA INVESTIGAÇÃO DA PROTEÍNA GRB2: EXPLORAÇÃO DA INTERAÇÃO ESPECÍFICA E ANÁLISE DINÂMICA DO ENOVELAMENTO Tese apresentada como parte dos requisitos para obtenção do título de Doutor em Biofísica Molecular, junto ao Programa de Pós Graduação em Biofísica Molecular do Instituto de Biociências, Letras e Ciências Exatas da Universidade Estadual Paulista “Júlio de Mesquita Filho”, Câmpus de São José do Rio Preto. Financiadora: CNPq - Processo: 140306/2020-0 Comissão Examinadora: Prof. Dr. Fernando Alves de Melo UNESP - Câmpus de São José do Rio Preto Orientador Prof. Dr João Ruggiero Neto UNESP - Câmpus de São José do Rio Preto Prof. Dr. Adolfo Henrique de Moraes Silva Universidade Federal de Minas Gerais - Belo Horizonte Prof. Dr. Luis Felipe Santos Mendes Universidade de São Paulo - São Carlos Prof. Dr. Roberto Kopke Salinas Universidade de São Paulo - São Paulo São José do Rio Preto 03 de abril de 2024 2 3 Este trabalho é dedicado a Ivanilda e Benedito, meus pais, cujo apoio constante sempre impulsionou meus sonhos. 3 4 AGRADECIMENTOS Primeiramente, expresso minha gratidão a Deus, pois reconheço que toda a trajetória é permeada por Sua orientação. Agradeço profundamente à minha família, em especial à minha mãe, Ivanilda, ao meu pai, Benedito, e ao meu irmão, Andrey, pelo apoio incondicional ao longo de toda a minha jornada. Sem o suporte de vocês, não teria alcançado este ponto crucial em minha vida acadêmica. Seu papel foi e continua sendo fundamental para o meu desenvolvimento pessoal e profissional. Estendo meus agradecimentos aos meus tios e avós, cuja ajuda e compreensão foram inestimáveis até aqui. Manifesto também minha gratidão à minha namorada, Isabela, por sua constante presença, apoio, compreensão e paciência ao longo desses anos. Ao meu orientador, Fernando, que tem sido um mentor desde os dias de graduação até o presente momento, expresso minha sincera gratidão. Sua orientação, respeito e paciência têm sido essenciais para o meu crescimento como profissional. Agradeço ao meu co-orientador, Ícaro, por sua disponibilidade em auxiliar e orientar, contribuindo significativamente para a minha formação. Expresso minha gratidão a todos os professores que, de diversas formas, contribuíram para minha formação acadêmica, em especial à professora Fátima, cuja abordagem diferenciada permitiu-me enxergar os desafios do cotidiano científico sob uma nova perspectiva. Ao Laboratório de Biofísica Molecular, onde adquiri as habilidades necessárias para a pesquisa, especialmente à Larissa, por suas contribuições científicas, momentos de descontração e discussões acadêmicas. Aos colegas do departamento de Física - Barbosa, Fernando, Isabela, Ingrid, João, Juliana, Karol, Larissa, Murilo, Raphael, Thainá e Thalita - expresso minha gratidão. Além de tornarem a jornada mais leve com sua amizade e bom humor, contribuíram significativamente para o meu crescimento acadêmico. Manifesto meu apreço à UNESP e ao IBILCE pela oportunidade de estudar em uma das melhores universidades do Brasil e do mundo, proporcionando-me oportunidades que jamais imaginei. Agradeço ao Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) pela concessão da bolsa de pesquisa, sob o processo nº 140306/2020-0. 4 5 “But still, like air, I’ll rise.” Maya Angelou em And Still I Rise (1). 5 https://www.zotero.org/google-docs/?L5INSP 6 RESUMO As células estão sujeitas a processos de regulação associados à sobrevivência, crescimento, diferenciação e apoptose. Durante o ciclo de proliferação celular, eventuais falhas podem resultar em um crescimento celular descontrolado, levando ao desenvolvimento de tumores. A via de sinalização MAPK é uma das vias impactadas por essas alterações, com a proteína adaptadora GRB2 desempenhando um papel significativo nesse contexto. A GRB2 é composta por três domínios, sendo um domínio SH2 flanqueado por dois domínios SH3 (N- e C-terminal). Embora as funções citoplasmáticas da GRB2 sejam bem conhecidas, uma nova função foi recentemente observada em vias de reparo de DNA. O reparo de quebras na fita dupla do DNA é iniciado pela proteína MRE11, direcionando a recombinação por homologia. Nesse processo, a proteína GRB2 atua como intermediária na interação entre o DNA e a MRE11, utilizando seu domínio SH2, ou seja, a proteína GRB2, através de se domínio SH2, reconhece a proteína MRE11 e a conduz para a região do DNA danificado e assim, então, iniciar a via de reparo. No entanto, até o momento, não houve investigações moleculares abordando essa interação. Com o objetivo de compreender a interação entre o domínio SH2 e o peptídeo MRE11, assim como possíveis interações com outras proteínas parceiras, foram conduzidos experimentos de 15N-HSQC-NMR. Durante a titulação entre o domínio SH2 e o peptídeo MRE11-pY, foram observadas consideráveis mudanças nos CSP dos resíduos de aminoácidos, especialmente no sítio de interação I, apresentando uma conformação inédita na literatura. Apesar de desafios de solubilidade, as análises de interação entre GRB2-SH2 e MRE11-22 indicaram uma interação significativa, conforme evidenciado por um CSP global. Experimentos de dinâmica molecular revelaram uma similaridade na forma de interação entre os peptídeos MRE11-pY e o MRE11-22. Tentativas de interação com um peptídeo curto não fosforilado, MRE11-07, não obtiveram sucesso. Experimentos adicionais com um peptídeo hipotético, MRE11-7Y, corroboraram os resultados obtidos para os peptídeos derivados de MRE11. Por fim, nas titulações entre GRB2-SH2 e peptídeos derivados da histona H2AX (H2AX-pSpY e H2AX-pS), não foram observadas perturbações químicas, indicando ausência de interação. No entanto, o peptídeo H2AX-pY demonstrou interação, sugerindo que a presença de uma fosfotirosina é crucial para a interação, enquanto a adição de uma fosfoserina impede tal interação. Esses resultados sugerem novas perspectivas para as funções da GRB2 em vias biológicas. Palavra-chave: GRB2, SH2, MRE11, H2AX, RMN. 6 7 ABSTRACT Cells are subject to regulatory processes associated with survival, growth, differentiation, and apoptosis. During the cell proliferation cycle, potential failures can result in uncontrolled cell growth, leading to tumor development. The MAPK signaling pathway is one of the pathways impacted by these alterations, with the adaptor protein GRB2 playing a significant role in this context. GRB2 is composed of three domains, with an SH2 domain flanked by two SH3 domains (N- and C-terminal). Although the cytoplasmic functions of GRB2 are well-known, a new function has recently been observed in DNA repair pathways. Repair of double-strand DNA breaks is initiated by the protein MRE11, directing homology recombination. In this process, the GRB2 protein acts as an intermediary in the interaction between DNA and MRE11, utilizing its SH2 domain. Specifically, the GRB2 protein, through its SH2 domain, recognizes the MRE11 protein and guides it to the region of damaged DNA to initiate the repair pathway. However, to date, there have been no molecular investigations addressing this interaction. To understand the interaction between the SH2 domain and the MRE11 peptide, as well as possible interactions with other partner proteins, 15N-HSQC-NMR experiments were conducted. During the titration between the SH2 domain and the MRE11-pY peptide, considerable changes in chemical shift perturbations of amino acid residues were observed, especially at interaction site I, presenting a novel conformation in the literature. Despite solubility challenges, interaction analyses between GRB2-SH2 and MRE11-22 indicated significant interaction, as evidenced by a global CSP. Molecular dynamics experiments revealed similarity in the interaction mode between MRE11-pY peptides and MRE11-22. Attempts to interact with a short unphosphorylated peptide, MRE11-07, were unsuccessful. Additional experiments with a hypothetical peptide, MRE11-7Y, corroborated the results obtained for peptides derived from MRE11. Lastly, in titrations between GRB2-SH2 and peptides derived from histone H2AX (H2AX-pSpY and H2AX-pS), no chemical shift perturbations were observed, indicating no interaction. However, the H2AX-pY peptide demonstrated interaction, suggesting that the presence of a phosphotyrosine is crucial for the interaction, while the addition of a phosphoserine impedes such interaction. These results suggest new perspectives for the functions of GRB2 in biological pathways. Keywords: GRB2, SH2, MRE11, H2AX, RMN. 7 8 LISTA DE FIGURAS Figura 1.1.1: Estrutura de um Receptor Tirosina-Quinase. 23 Figura 1.1.2: Representação em cartoon da proteína GRB2 em sua configuração monomérica. 24 Figura 1.1.3: Vias de Sinalização Mediadas pela Proteína GRB2. 27 Figura 1.1.4: Modelo do Recrutamento do Complexo MRN em Quebras de Fita Dupla (DSBs). 30 Figura 1.1.5: Configuração tridimensional do domínio SH2 da proteína GRB2. 32 Figura 1.1.6: Domínio SH2 das Proteínas LNK e GRB2. 33 Figura 1.2.1: Momento Magnético Nuclear. 37 Figura 1.2.2: Representação dos Estados de Spin Nuclear. 38 Figura 1.2.3: Precessão de Larmor. 39 Figura 1.2.4: Vetor de Magnetização Bulk. 41 Figura 1.2.5: Acoplamento de Spin Nuclear em um Grupo 13C-H. 43 Figura 1.2.6: Representação Vetorial da Variação da Magnetização ao Longo de um Experimento de HSQC. 47 Figura 1.2.7: Dependência do Pico de RMN Bidimensional com a Taxa de Troca. 53 Figura 1.2.8: Algoritmos de pesquisa e métricas de pontuação empregados em docking molecular. 56 Figura 1.2.9: Os elementos de um campo de força, que denotam interações vinculadas e não vinculadas. 63 Figura 1.4.1: Diagrama do vetor pET-28a(+). 71 Figura 1.5.1: Purificação do domínio GRB2-SH2. 80 Figura 1.5.2: Análise dos principais CSP na interação entre GRB2-SH2 e MRE11-pY. 82 Figura 1.5.3: Titulação de MRE11-pY no domínio GRB2-SH2 monitorado por experimento de HSQC. 84 Figura 1.5.4: RMSD da interação entre o domínio GRB2-SH2 e o peptídeo MRE11-pY. 85 8 9 Figura 1.5.5: Representação em cartoon do complexo GRB2-SH2 ligado ao fosfopeptídeo MRE11-pY, acompanhado de uma visualização esquemática da interação através do LigPlot. 87 Figura 1.5.6: Análise dos CSP mais proeminentes na interação entre GRB2-SH2 e MRE11-22. 89 Figura 1.5.7: Titulação de MRE11-22 no domínio GRB2-SH2 monitorado por experimento de HSQC. 91 Figura 1.5.8: Análise da dinâmica molecular entre o domínio GRB2-SH2 e o peptídeo MRE11-22. 92 Figura 1.5.9: Representação em cartoon do domínio GRB2-SH2 complexado com o peptídeo MRE11-22. 93 Figura 1.5.10: Titulação de MRE11-07 no domínio GRB2-SH2 monitorado por experimento de HSQC. 95 Figura 1.5.11: Representação em cartoon da interação dos fosfopeptídeos derivado de EGFR e de MRE11. 97 Figura 1.5.12: Representação em cartoon da interação dos peptídeos derivados de MRE11. 99 Figura 1.5.13: Titulação dos fosfopeptídeos derivados de H2AX no domínio GRB2-SH2 monitorada por meio do experimento de HSQC. 101 Figura 1.5.14: Análise dos principais CSP na interação entre GRB2-SH2 e H2AX-pY. 103 Figura 1.5.15: Titulação de MRE11-pY no domínio GRB2-SH2 monitorado por experimento de HSQC. 105 Figura 1.5.16: Análise da dinâmica molecular entre o domínio GRB2-SH2 e o peptídeo H2AX-pY. 106 Figura 1.5.17: Representação em cartoon do complexo GRB2-SH2 ligado ao fosfopeptídeo H2AX-pY, acompanhado de uma representação esquemática da interação através do LigPlot. 108 Figura 1.5.18: Representação em cartoon da interação dos fosfopeptídeos derivado de EGFR e de H2AX. 109 Figura 2.5.1: Processo de Purificação da Proteína GRB2. 123 Figura 2.5.2: Análise do espectro de emissão de fluorescência da proteína GRB2 em variações de temperatura. 124 Figura 2.5.3: Determinação do Centro de Massa Espectral (CM). 125 9 10 Figura 2.5.4: Termograma da Proteína GRB2 obtido por meio de Calorimetria Diferencial de Varredura. 126 Figura 2.5.5: Análises Termodinâmicas dos Perfis da Proteína GRB2 e do Domínio GRB2-SH2 Conduzidas por Calorimetria Diferencial de Varredura (DSC). 127 Figura 2.5.6: Correlação Estrutural entre o Funil de Energia e os Mapas de Contato é Destacada, Evidenciando os Conjuntos Correspondentes a cada Mapa. 129 Figura SIV.1: Resíduos com chemical shift mais proeminentes da interação entre o domínio GRB2-SH2 e o peptídeo MRE11-pY. 147 Figura SV.1: Resíduos com chemical shift mais proeminentes da interação entre o domínio GRB2-SH2 e o peptídeo MRE11-22. 154 Figura SVI.1: Resíduos com chemical shift mais proeminentes da interação entre o domínio GRB2-SH2 e o peptídeo H2AX-pY. 164 10 11 LISTA DE TABELAS Tabela 1.1.1: Exemplos de Proteínas Receptoras: 21 Tabela 1.1.2: Exemplos de proteínas sinalizadoras e receptores tirosina-quinases (RTKs). 22 Tabela 1.2.1: Características dos Isótopos mais Empregados em Ressonância Magnética Nuclear. 36 Tabela 1.2.2: Estatísticas do Protein Data Bank. 61 Tabela 1.4.1: Peptídeos Utilizados no Estudo. 75 Tabela 1.4.2: Relação dos experimentos de HSQC das titulações dos peptídeos. 76 Tabela 1.5.1: Propriedades físico-químicas teóricas. 79 Tabela 1.5.2: Resíduos que apresentaram CSP maiores que a média e maiores que a média mais desvio padrão. 83 Tabela 1.5.3: Resíduos que apresentaram CSP maiores que a média e maiores que a média mais desvio padrão. 90 Tabela 1.5.4: Resíduos mais proeminentes na interação GRB2-SH2/MRE11-pY e GRB2-SH2/MRE11-22. 94 Tabela 1.5.5: Resíduos que demonstraram CSP superiores à média e que excederam a média acrescida do desvio padrão. 104 Tabela 2.5.1: Características Teóricas de Natureza Físico-Química da Proteína GRB2. 122 Tabela AI.1: Solução RF1. 142 Tabela AI.2: Solução RF2. 142 Tabela AI.3: Meio SOB. 142 Tabela AVII.1: Pré-meio mínimo de 200 ml. 168 Tabela AVII.2: Pré-meio mínimo de 1000 ml. 169 11 12 LISTA DE ABREVIATURAS E SIGLAS AMBER Assisted Model Building with Energy Refinement BER Reparo por Excisão de Bases BMRB Biological Magnetic Resonance data Bank CHARMM Chemistry at HARvard Macromolecular Mechanics CSP Perturbação do Deslocamento Químico DDR Resposta a Danos no DNA DMSO Dimetilsulfóxido DNA Ácido Desoxirribonucleico DSB Quebras da dupla-hélice de DNA EGFR Epidermal Growth Factor Receptor FGFR2 Fibroblast Growth Factor Receptor 2 FGFs Fatores de Crescimento de Fibroblastos GDP Guanosina difosfato GRB2 Growth Factor Receptor-Bound protein 2 GTP Guanosina-5'-trifosfato HDR Recombinação dirigido por Homologia HSQC Heteronuclear Single Quantum Correlation INEPT Insensitive Nuclei Enhanced by Polarization Transfer MAPK Mitogen-Activated Protein Kinase MMR Reparo de Mau-pareamento MRE11 Meiotic Recombination 11 homolog MRN Complexo formado pelas proteínas MRE11, RAD50 e NBS1 NER Reparo por Excisão de Nucleotídeos NHEJ União de Extremidades não Homólogas PDB Protein Data Bank PI3K Phosphoinositide 3-kinases 12 13 PTEN Phosphatase and Tensin Homolog RAS Rat sarcoma virus RMN Ressonância Magnética Nuclear RMSD Root Mean Square Deviation RMSF Root Mean Square Fluctuation RTKs Receptores Tirosinas-Quinases SDS-PAGE Eletroforese em Gel de Poliacrilamida com Dodecilsulfato de Sódio SH2 Src Homology 2 SH3 Src Homology 3 SHP2 Src homology region 2 domain-containing phosphatase-2 SOS Son of Sevenless 13 14 SUMÁRIO 1. INTRODUÇÃO....................................................................................................................16 2. CAPÍTULO 1.......................................................................................................................18 2.1. INTRODUÇÃO.................................................................................................... 19 2.1.1. SINALIZAÇÃO CELULAR...........................................................................19 2.1.2. A PROTEÍNA GRB2 E VIAS DE SINALIZAÇÃO........................................ 24 2.1.3. A PROTEÍNA GRB2, A PROTEÍNA MRE11 E A VIA DE REPARO DE DNA. 28 2.1.4. O DOMÍNIO SH2 DA PROTEÍNA GRB2.................................................... 31 2.2. FUNDAMENTAÇÃO TEÓRICA.......................................................................................36 2.2.1. FUNDAMENTOS BÁSICOS DE ESPECTROSCOPIA DE RESSONÂNCIA MAGNÉTICA NUCLEAR.......................................................................................36 2.2.1.1. Acoplamento J....................................................................................41 2.2.1.2. 15N, 1H-HSQC (Heteronuclear Single Quantum Correlation)....................... 45 2.2.1.3. Perturbação do Deslocamento Químico........................................................ 48 2.2.2. DOCKING MOLECULAR............................................................................55 2.2.3. DINÂMICA MOLECULAR........................................................................... 60 2.2.3.1. Princípios da Dinâmica Molecular..................................................................62 2.2.3.2. Condições Periódicas de Contorno................................................................64 2.2.3.3. Termostato e Modelos de Solventes.............................................................. 65 2.2.3.4. Minimização de Energia em Simulações de Dinâmica Molecular..................65 2.2.3.5. Análises RMSD e RMSF................................................................................67 2.3. OBJETIVOS.................................................................................................................... 70 2.3.1. OBJETIVOS ESPECÍFICOS.......................................................................70 2.4. METODOLOGIA..............................................................................................................71 2.4.1. CONSTRUÇÃO DO PLASMÍDEO.............................................................. 71 2.4.2. TRANSFORMAÇÃO DO PLASMÍDEO RECOMBINANTE EM CÉLULAS DE ESCHERICHIA COLI LINHAGEM DH5α........................................................ 72 2.4.3. TRANSFORMAÇÃO EM CÉLULAS DE ESCHERICHIA COLI LINHAGEM BL21 (DE3)........................................................................................................... 73 2.4.4. EXPRESSÃO E PURIFICAÇÃO DO DOMÍNIO 15N-GRB2-SH2...............73 2.4.5. PEPTÍDEOS DERIVADOS DE MRE11 E H2AX..................................................... 74 2.4.6. ESPECTROSCOPIA DE ABSORÇÃO UV-VIS...........................................75 2.4.7. ESPECTROSCOPIA DE RESSONÂNCIA MAGNÉTICA NUCLEAR.........75 2.4.8. DOCKING E DINÂMICA MOLECULAR..................................................................77 2.5. RESULTADOS E DISCUSSÕES.....................................................................................79 2.5.1. PARÂMETROS FÍSICO-QUÍMICOS DO DOMÍNIO GRB2-SH2 E DOS PEPTÍDEOS..........................................................................................................79 2.5.2. EXPRESSÃO E PURIFICAÇÃO DO DOMÍNIO 15N-GRB2-SH2...............79 2.5.3. INTERAÇÃO ENTRE MRE11-pY E GRB2-SH2......................................... 81 2.5.4. INTERAÇÃO ENTRE MRE11-22 E GRB2-SH2..........................................88 15 2.5.5. INTERAÇÃO ENTRE MRE11-07 E GRB2-SH2..........................................94 2.5.6. MODO DE INTERAÇÃO ENTRE OS PEPTÍDEOS DERIVADOS DE MRE11 E O DOMÍNIO GRB2-SH2....................................................................................................... 96 2.5.7. INTERAÇÕES ENTRE H2AX-pSpY E GRB2-SH2 E ENTRE H2AX-pS E GRB2-SH2.......................................................................................................... 100 2.5.8. INTERAÇÃO ENTRE H2AX-pY E GRB2-SH2......................................................102 2.6. CONCLUSÕES..............................................................................................................110 2.7. PERSPECTIVAS............................................................................................................113 3. CAPÍTULO 2.....................................................................................................................114 3.1. PREÂMBULO E CONTEXTUALIZAÇÃO..................................................................... 115 3.2. INTRODUÇÃO...............................................................................................................116 3.3. OBJETIVOS...................................................................................................................119 3.3.1. OBJETIVOS ESPECÍFICOS................................................................................. 119 3.4. METODOLOGIA............................................................................................................120 3.4.1. PREPARO DE PROTEÍNA................................................................................... 120 3.4.2. ESPECTROSCOPIA DE FLUORESCÊNCIA....................................................... 120 3.4.3. ANÁLISE TÉRMICA POR CALORIMETRIA DIFERENCIAL DE VARREDURA...121 3.5. RESULTADOS E DISCUSSÕES...................................................................................122 3.5.1. PARÂMETROS FÍSICO-QUÍMICOS DA PROTEÍNA GRB2.................................122 3.5.2. EXPRESSÃO E PURIFICAÇÃO DA PROTEÍNA GRB2.......................................122 3.5.3. PERFIL DE DESENOVELAMENTO DA PROTEÍNA GRB2 VIA FLUORESCÊNCIA. 123 3.5.4. PERFIL DE DESENOVELAMENTO DA PROTEÍNA GRB2 VIA CALORIMETRIA DE VARREDURA DIFERENCIAL................................................................................... 126 3.6. CONCLUSÕES..............................................................................................................130 3.7. PERSPECTIVAS........................................................................................................... 131 4. CONSIDERAÇÕES FINAIS............................................................................................. 132 REFERÊNCIAS........................................................................................................133 ANEXO A................................................................................................................. 143 ANEXO B................................................................................................................. 145 ANEXO C................................................................................................................. 146 ANEXO D................................................................................................................. 148 ANEXO E................................................................................................................. 155 ANEXO F.............................................................................................................................. 165 ANEXO G.................................................................................................................169 ANEXO H..............................................................................................................................171 16 1. INTRODUÇÃO A proteína Growth Factor Receptor-Bound 2 (GRB2) é um componente chave nas vias de sinalização intracelular, desempenhando um papel crucial na transmissão de sinais extracelulares para o interior da célula. Sua importância é evidenciada pela sua conservação evolutiva em organismos, destacando sua função fundamental em processos fisiológicos essenciais. A estrutura da GRB2 compreende diversos domínios funcionais, incluindo um domínio N-terminal SH3, um domínio central SH2 e um domínio C-terminal SH3. Estes domínios conferem à GRB2 a capacidade de interagir com múltiplos parceiros moleculares dentro da célula, desempenhando papéis variados em diferentes contextos celulares. A principal função conhecida da GRB2 é sua participação em vias de sinalização de receptores tirosina-quinase (RTKs), onde atua como um mediador entre o receptor ativado e a proteína SOS, desencadeando a ativação da proteína Ras e subsequente ativação de cascatas de sinalização, como a via MAPK. Além disso, a GRB2 também está envolvida em outras vias de sinalização, como a via de sinalização ativada por integrinas e a via de sinalização mediada por citocinas e mais recentemente vias de reparo de DNA. No contexto da regulação do ciclo celular e da diferenciação celular, a GRB2 desempenha papéis cruciais, influenciando a proliferação celular, a sobrevivência e a migração. Alterações na expressão ou na atividade da GRB2 foram associadas a diversas patologias, incluindo câncer, diabetes e doenças cardiovasculares, destacando a importância de entendermos os mecanismos de regulação dessa proteína em condições normais e patológicas. Considerando sua importância central nas vias de sinalização celular e seu potencial como alvo terapêutico, a proteína GRB2 é objeto de estudo em diversos campos da biologia e da medicina. No contexto específico desta tese de doutorado, a GRB2 será investigada em dois capítulos distintos. No primeiro capítulo, será examinada a interação entre a GRB2 e peptídeos relevantes para a via de reparo de DNA, proporcionando conhecimentos sobre seu papel na manutenção da integridade genômica. No segundo capítulo, serão explorados os mecanismos de enovelamento da GRB2, ampliando nossa compreensão sobre sua estrutura e dinâmica molecular. Esses estudos contribuirão para uma melhor compreensão dos 17 processos biológicos regulados pela GRB2 e para o desenvolvimento de estratégias terapêuticas mais eficazes no contexto de doenças relacionadas. 132 4. CONSIDERAÇÕES FINAIS A análise das interações entre proteínas e peptídeos é essencial para a compreensão dos mecanismos de ação das proteínas nas vias biológicas, fornecendo uma perspectiva molecular do ambiente de ligação e seu impacto na função proteica. Este estudo investigou a interação entre o domínio GRB2-SH2 e os peptídeos MRE11-pY, MRE11-22 e MRE11-07, derivados da proteína MRE11, bem como os fosfopeptídeos H2AX-pSpY, H2AX-pS e H2AX-pY, derivados da histona H2AX. No caso do MRE11-pY, foi observada uma interação não convencional, com a fosfotirosina se ligando ao sítio de interação I, enquanto os resíduos pY+1 a pY+5 permaneceram fora do sítio II. O peptídeo MRE11-22 exibiu um comportamento semelhante ao peptídeo MRE11-pY. Experimentos com o MRE11-07 não revelaram interações, atribuídas à escassez de resíduos e à ausência de tirosina fosforilada. Quanto aos fosfopeptídeos H2AX-pSpY e H2AX-pS, não foram observadas perturbações significativas nos deslocamentos químicos, sugerindo uma possível competição entre fosfotirosina e fosfoserina ou um efeito inibitório da fosfoserina na interação com o domínio SH2. 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